1. Field of the Invention
The present invention relates to a method and apparatus for indirect agglutination immunoassay in which agglutination being caused by an antigen-antibody reaction is assayed using magnetic particles or magnetic-material-containing particles that have an antigen or antibody bound thereto.
2. Description of the Related Art
The inventors of the present invention previously proposed an effective method and apparatus for indirect agglutination immunoassay as U.S. patent application Ser. No. 08/082,373 "INDIRECT AGGLUTINATION IMMUNOASSAY AND APPARATUS THEREFOR" filed on Jun. 28, 1993 which is a Continuation-in-Part Application of U.S. patent application Ser. No. 07/606,205 filed on Oct. 31, 1990. According to the method, a solution of the particles of a specified reagent is added and mixed with a diluted solution of a specified sample in each of wells with a U- or V-shaped bottom in a micro plate and, after some reaction, components including the particles are precipitated in the bottom of each well by magnetic force and, subsequently, the micro plate is inclined to judge the occurrence of an immunoreaction in view of the profile of separation of the precipitated particles from the wells.
The precipitation patterns that are to be formed in the method of indirect agglutination immunoassay due to the flow of the particles in the micro plate are roughly divided into three types: i) a reaction-positive precipitation pattern that results from the bonding among particles due to an immunoreaction and which is characterized by stationary particles that do not move but which remain as a spot in the bottom of each well even if the micro plate is inclined; ii) a reaction-negative precipitation pattern that results from the non-binding among particles due to the absence of an immunoreaction and which is characterized by free-flowing particles that, upon inclination of the micro plate, flow down to the side wall of each well; and iii) a precipitation pattern intermediate between i) and ii).
The precipitation patterns due to the flow of the particles that are formed in the bottoms of wells in a micro plate can be read by various methods such as taking a picture of the micro plate from above with a TV camera or, alternatively, using a line sensor and taking a picture of the precipitation patterns due to the flow of the particles by image processing techniques for measuring the flow lengths of the respective patterns due to the flow of the particles so that they can be correlated to the occurrence or non-occurrence of an immunoreaction.
The method of indirect agglutination immunoassay filed as the U.S. patent application Ser. No. 08/082,373 is efficient in that it is capable of judging the occurrence of an immunoreaction in a shorter time and with higher precision than other approaches such as a standing method in which particles are allowed to precipitate under gravity within wells in a micro plate and in which judgement for the occurrence of an antigen-antibody reaction is made on the basis of the precipitation pattern of the particles. However, in the practice of this method, it is essential that the precipitation patterns due to the flow of the particles be read by a TV camera, a line sensor or the like.
A major problem with the methods of picking up image with a TV camera, a line sensor or the like is that the distance which have to be provided between the TV camera, line sensor or the like and the micro plate makes it imperative to use a large device for judging precipitation patterns; if the distance between the two elements is unduly short, particularly in the case where the micro plate has U- or V-shaped wells, the distance to the TV camera, line sensor or the like is not invariable in the center of the bottom of each well and its peripheral portion and, hence, defocusing occurs to cause disadvantages such as distorted image.
Glass or synthetic resin (e.g., polystyrene) micro plate used in indirect agglutination immunoassay has in most cases a plurality of wells with U- or V-shaped bottoms and the micro plate has ninety six wells (in 12 columns .times.8 rows). When molding micro plates, the wells which are formed to have U- or V-shaped bottoms may unavoidably experience molding distortion at the edges on the periphery of well's bottom and, hence, the wells formed may not necessarily lie on one straight line in the direction of columns or rows. Accordingly, even if the sensor is moved along the central part of the bottom of each well in the direction of either columns or rows after the formation of precipitation patterns, the above-mentioned misalignment introduces inaccuracy in the measurement of the length of precipitation patterns.
Thus, the misalignment in the wells that are formed by molding a micro plate reduces the precision in the measurement of precipitation patterns due to the flow of the particles which provide criteria for the occurrence of an immunoreaction, is the correct judgement for the occurrence of an immunoreaction provided and the reproducibility of data is fluctuated greatly.
In addition, irrespective of the V- or U-shape micro plate is used to judge for the occurrence of an immunoreaction by indirect agglutination techniques, precipitation patterns due to the flow of particles are formed on slopes that extend from the centers of the bottoms of respective wells.
Therefore, when the lengths of precipitation patterns due to the flow of the particles formed on the slopes of the wells are to be read optically with a sensor or the like, a defocusing problem may occur since the distance to the sensor differs between the center of each well and its periphery.